Since the early 1970's when routine chromosome banding was developed, Giemsa-banded chromosome analysis has been applied to diagnosing chromosome abnormalities in fetuses, abnormal children, adolescents, and adults, in both normal and neoplastic tissues. Giemsa-banded karyotypes will detect abnormal chromosomes in about 644 newborns among every 100,000 births (Lebo et al, 1992). Banded chromosome analysis is time consuming and requires considerable training and expertise from growing the cells and preparing slides of well separated, banded chromosomes, to recognizing and analyzing spreads of randomly mixed metaphase banded chromosomes from selected cells for whole and partial chromosome abnormalities. Nevertheless, chromosome banding identifies only about half of all genetic abnormalities because the limit of light microscope resolution is on the order of 5,000,000 basepairs of DNA (5 Mb spanning an average of 50 genes) that must be modified in order to detect a change in the chromosome banding pattern. In contrast, molecular testing can use sampled cells that have not grown outside the body, complete analysis in hours rather than days, and distinguish the modification of a single basepair change or quantify the number of target gene sequences that may have changed within a normal appearing banded chromosome. With the exception of chromosome banding, a single format has not been applied successfully to genome-wide screening.
Initially we conceived and developed a screening test for aneuploidy of five chromosomes (13, 18, 21, X, and Y) that result in 95% of chromosomally abnormal newborns (Lebo et al, 1992). This test has been modified by other investigators to enumerate chromosome 13 and chromosome 21 independently and with simultaneous commercialization and wider testing validation by Vysis has received FDA approval. Today this is used for late gestation fetuses to determine rapidly whether a fetus with an abnormal ultrasound has one of these viable chromosome aneuploidies in order to optimally plan delivery (Lapidot-Lifson et al, 1996) and to obtain a rapid result for earlier gestation pregnancies undergoing triple screen analysis. G-banded karyotypes are still completed routinely on all sampled fetal cells (amniocytes or chorionic villus cells).
Considering these developments, our initial patent application suggested selecting carefully chosen genome-wide chromosome sites to be tested for aneuploidy in order to detect the largest proportion of chromosome rearrangements resulting in partial or full chromosome aneuploidy, and to test for all additional submicroscopic and microscopic deletions that commonly result in genetic disease because this would be a more rapid test that detected a larger number of abnormal fetuses than Giemsa-banded karyotyping (Lebo et al., Provisional 60/161857). As we have continued to work on this approach, we designated the most common gene mutations to be tested simultaneously to detect the largest number of genetic abnormalities possible in a single test on a minimal size testing format.
More recently Snijders et al., (2000) applied CGH to segments of chromosomes at 1 Mb regions in order to detect aneuploid (absence of two) copies of each location reflecting chromosome rearrangement. This requires >2,000 sites to test the 3,000,000,000 basepair haploid human genome at ˜1 megabase intervals. Two difficulties were not anticipated using this approach: (1) the greater the number of sites tested, the greater the likelihood that an error will occur given the same error frequency at each tested site, and (2) tested sites were designated according to physical distance rather than selecting genetically important sites that when mutated result in the most common disease-causing mutations. Thus a large proportion of normal patients tested at these >2000 sites have deleted chromosome regions that merely reflect normal polymorphic variability (Alfred Mazzocchi, Vysis Molecular specialist-Midwest, Pers. Comm., August, 2002). Therefore this approach requires determining the normal polymorphic variability in the general population and the restructuring of the sites selected.
The cystic fibrosis gene is mutated by any one of over 1000 mutations carried by 1 in 29 Caucasians. Over two dozen laboratories offer routine cystic fibrosis testing for 12 to 100 cystic fibrosis mutations. The number of mutation tests offered reflect not only the frequency each mutation is found within the tested population but also differences in the laboratory's prior experience in identifying specific cystic fibrosis mutations, and the likelihood of test referral from genetics professionals based upon the number of tested mutations. The economic principle of “diminishing returns” states that when any factor is increased while other factors are held constant in amount, the gain in benefit beyond a certain point will diminish for each additional unit of resources invested. Given an ever larger number of mutations tested and an equal probability of error on each single mutation test provided, the probability of laboratory error could exceed the likelihood of finding any tested mutation. Given that most cystic fibrosis mutations are extremely rare and the likelihood of making a laboratory error may exceed the likelihood of finding a rare mutation, the American College of Medical Genetics committee on cystic fibrosis testing decided that testing the 25 mutations found in >0.1% of the cystic fibrosis mutant alleles in all Caucasions is to be considered standard-of-care for all testing laboratories. Selecting these 25 mutations opened the opportunity for the best laboratories to test other common disease gene mutations that detect many more abnormal alleles than tests for very rare alleles at one gene site. Reflex gene mutation or sequencing tests provide the opportunity to complete the most reliable diagnoses in higher-risk patient populations.
The following references are relevant as background to the present invention:    Lebo R V, Saiki R K, Swanson K, Montano M A, Erlich H A, Golbus M S: Prenatal diagnosis of α-thalassemia by PCR and dual restriction enzyme analysis. Hum Genet 85:293-299, 1990.    Lebo R V, Lynch E D, Golbus M S, Yen P H, Shapiro L: Prenatal in situ hybridization test for deleted steroid sulfatase gene. Am J Med Genet 46(6):652-658, 1993a.    Lebo R V, Martelli L, Su Y, Li L-Y, Lynch E, Mansfield E, Pua K, Watson D, Chueh J, Hurko O: Prenatal diagnosis of Charcot-Marie-Tooth disease Type 1A by multicolor in situ hybridization. Am J Med Genet 47(3):441-450, 1993b.    Mansfield E S. Diagnosis of Down syndrome and other aneuploidies using quantitative polymerase chain reaction and small tandem repeat polymorphisms. Hum Molec Genet 1992; 2:43-50.    Pinkel D, Albertson D, Gray J W, Comparative fluorescence hybridization to nucleic acid arrays. U.S. Pat. No. 5,830,645. Nov. 3, 1998.    Riordan et al., “Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066-1073, 1989.    Snijders A M, Hindle A K, Segraves R, Blackwood S, Myambo K, Yue P, Zhang X, Hamilton G., Brown N, Huey B, Law S, Gray J, Pinkel D, Albertson D G. Quantitative DNA copy number analysis across the human genome with ˜1 megabase resolution using array CGH. Am J Hum Genet 67(4) 31, 2000.    Wyandt H, Lebo R, Yosunkawa Fenerci E, Sadhu D N, Milunsky J. Molecular and cytogenetic characterization of duplication/deletion in a supernumerary der(9) resulting in 9p trisomy and partial 9q tetrasomy. Am J Med Genet 93:305-312, 2000.    Lebo R V, Flandermeyer R R, Lynch E D, Lepercq J A, Diukman R, Golbus M: Prenatal diagnosis with repetitive in situ hybridization probes. Am J Med Genet 43:848-854, 1992.    Gardner R J M and Sutherland G R. Chromosome Abnormalities and Genetic Counseling. Oxford Monographs on Medical Genetics No. 29, Oxford University Press, 1996, pp. 87-89.    Milunsky J M, Lebo R V, Ikuta T, Maher T A, Haverty C E, Milunsky A. Mutation Analysis in Rett Syndrome. Genetic Testing 5(4):321-325, 2001.    Herbergs J, Smeets E, Moog U, Tserpelis D, Smeets H. MECP2 mutation analysis and genotype/phenotype correlation in 26 Dutch Rett syndrome patients. Am J Hum Genet 69(4):306, 2001.    Lebo R V, Flandermeyer R R, Lynch E D, Lepercq J A, Diukman R, Golbus M: Prenatal diagnosis with repetitive in situ hybridization probes. Am J Med Genet 43:848-854, 1992.    Milunsky J M, Lebo R V, Ikuta T, Maher T A, Haverty C E, Milunsky A. Mutation Analysis in Rett Syndrome. Genetic Testing 5(4):321-325, 2001.